VISTA Antagonist and Methods of Use

ABSTRACT

The present invention is directed to a peptide, multimer, conjugate, analog, derivative or mimetic thereof that inhibits the activity of VISTA. The invention further contemplates therapeutic use of the VISTA antagonist peptide, multimer, conjugate, derivative or mimetic thereof, including treating or preventing cancer, bacterial infections, viral infections, parasitic infections and fungal infections, as well as research uses of the antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 13/925,094, filedJun. 24, 2013, which claims priority to U.S. Provisional Ser. No.61/663,969, filed Jun. 25, 2012, and U.S. Provisional Ser. No.61/663,431, filed Jun. 22, 2012, and also claims priority to U.S.Provisional Ser. No. 61/927,061, filed on Jan. 14, 2014, the contents ofeach, including the sequence listing, are incorporated herein byreference in their entireties.

The sequence listing file named “43260o1003”, having a size of 688 bytesand created Nov. 6, 2014, is hereby incorporated by reference in itsentirety.

BACKGROUND

The immune system is tightly controlled by co-stimulatory andco-inhibitory ligands and receptors. These molecules provide not only asecond signal for T cell activation but also a balanced network ofpositive and negative signals to maximize immune responses againstinfection while limiting immunity to self.

Induction of an immune response requires T cell expansion,differentiation, contraction and establishment of T cell memory. T cellsmust encounter antigen presenting cells (APCs) and communicate via Tcell receptor (TCR)/major histocompatibility complex (MHC) interactionson APCs. Once the TCR/MHC interaction is established, other sets ofreceptor-ligand contacts between the T cell and the APC are required,i.e. co-stimulation via CD154/CD40 and CD28/B7.1-B7.2. The synergybetween these contacts results in a productive immune response capableof clearing pathogens and tumors, and may be capable of inducingautoimmunity.

Another level of control has been identified, namely regulatory T cells(T_(reg)). This specific subset of T cells is generated in the thymus,delivered into the periphery, and is capable of constant and induciblecontrol of T cells responses. Sakaguchi (2000) Cell 101(5):455-8;Shevach (2000) Annu. Rev. Immunol. 18:423-49; Bluestone and Abbas (2003)Nat. Rev. Immunol. 3(3):253-7. T_(reg) are represented by a CD4+CD25+phenotype and also express high levels of cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4), OX-40, 4-1BB and theglucocorticoid inducible TNF receptor-associated protein (GITR). McHugh,et al. (2002) Immunity 16(2):311-23; Shimizu, et al. (2002) Nat. Immun.3(2):135-42. Elimination of T_(reg) cells by 5 day neonatal thymectomyor antibody depletion using anti-CD25, results in the induction ofautoimmune pathology and exacerbation of T cells responses to foreignand self-antigens, including heightened anti-tumor responses. Sakaguchi,et al. (1985) J. Exp. Med. 161(1):72-87; Sakaguchi, et al. (1995) J.Immunol. 155(3):1151-64; Jones, et al. (2002) Cancer Immun. 2:1. Inaddition, T_(reg) have also been involved in the induction andmaintenance of transplantation tolerance, since depletion of T_(reg)with anti-CD25 monoclonal antibodies results in ablation oftransplantation tolerance and rapid graft rejection. Jarvinen, et al.(2003) Transplantation 76:1375-9. Among the receptors expressed byT_(reg) GITR seems to be an important component since ligation of GITRon the surface of Treg with an agonistic monoclonal antibody results inrapid termination of T,_(g) activity, resulting in autoimmune pathologyand ablation of transplantation tolerance.

Costimulatory and co-inhibitory ligands and receptors not only provide a“second signal” for T cell activation, but also a balanced network ofpositive and negative signal to maximize immune responses againstinfection while limiting immunity to self. The best characterizedcostimulatory ligands are B7.1 and B7.2, which are expressed byprofessional APCs, and whose receptors are CD28 and CTLA-4. Greenwald,et al. (2005) Annu Rev Immunol 23, 515-548; Sharpe and Freeman (2002)Nat Rev Immunol 2, 116-126. CD28 is expressed by naïve and activated Tcells and is critical for optimal T cell activation. In contrast, CTLA-4is induced upon T cell activation and inhibits T cell activation bybinding to B7.1/B7.2, thus impairing CD28-mediated costimulation. CTLA-4also transduces negative signaling through its cytoplasmic ITIM motif.Teft, et al. (2006). Annu Rev Immunol 24, 65-97. B7.1/B7.2 KO mice areimpaired in adaptive immune response (Borriello, et al. (1997) Immunity6, 303-313; Freeman, et al. (1993) Science 262, 907-909), whereas CTLA-4KO mice can not adequately control inflammation and develop systemicautoimmune diseases. Chambers, et al. (1997) Immunity 7, 885-895; Tivol,et al. (1995) Immunity 3, 541-547; Waterhouse, et al. (1995) Science270, 985-988. The B7 family ligands have expanded to includecostimulatory B7-H2 (ICOS Ligand) and B7-H3, as well as co-inhibitoryB7-H1 (PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), and B7-H6. SeeBrandt, et al. (2009) J Exp Med 206, 1495-1503; Greenwald, et al. (2005)Annu Rev Immunol 23: 515-548.

Inducible costimulatory (ICOS) molecule is expressed on activated Tcells and binds to B7-H2. See Yoshinaga, et al. (1999) Nature 402,827-832. ICOS is important for T cell activation, differentiation andfunction, as well as essential for T-helper-cell-induced B cellactivation, Ig class switching, and germinal center (GC) formation.Dong, et al. (2001) Nature 409, 97-101; Tafuri, et al. (2001) Nature409, 105-109; Yoshinaga, et al. (1999) Nature 402, 827-832. ProgrammedDeath 1 (PD-1) on the other hand, negatively regulates T cell responses.PD-1 KO mice develop lupus-like autoimmune disease, or autoimmunedilated cardiomyopathy depending upon the genetic background. Nishimura,et al. (1999) Immunity 11, 141-151. Nishimura, et al. (2001) Science291: 319-322. The autoimmunity most likely results from the loss ofsignaling by both ligands PD-L1 and PD-L2. Recently, CD80 was identifiedas a second receptor for PD-L1 that transduces inhibitory signals into Tcells. Butte, et al. (2007) Immunity 27: 111-122. The receptor for B7-H3and B7-H4 still remain unknown.

The best characterized co-stimulatory ligands are B7.1 and B7.2, whichbelong to the Ig superfamily and are expressed on professional APCs andwhose receptors are CD28 and CTLA-4 (Greenwald, et al. (2005) Annu. Rev.Immunol. 23:515-548). CD28 is expressed by naïve and activated T cellsand is critical for optimal T cell activation. In contrast, CTLA-4 isinduced upon T cell activation and inhibits T cell activation by bindingto B7.1/B7.2, impairing CD28-mediated co-stimulation. B7.1 and B7.2 KOmice are impaired in adaptive immune response (Borriello, et al. (1997)Immunity 6:303-313), whereas CTLA-4 knockout mice cannot adequatelycontrol inflammation and develop systemic autoimmune diseases (Tivol, etal. (1995) Immunity 3:541-547; Waterhouse, et al. (1995) Science270:985-988; Chambers, et al. (1997) Immunity 7:885-895).

The B7 family ligands have expanded to include co-stimulatory B7-H2(inducible T cell co-stimulator (ICOS) ligand) and B7-H3, as well asco-inhibitory B7-H1 (PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), andB7-H6 (Greenwald, et al. (2005) supra; Brandt, et al. (2009) J. Exp.Med. 206:1495-1503). Accordingly, additional CD28 family receptors havebeen identified. ICOS is expressed on activated T cells and binds toB7-H2 (Yoshinaga, et al. (1999) Nature 402:827-832). ICOS is a positivecoregulator, which is important for T cell activation, differentiation,and function (Yoshinaga, et al. (1999) supra; Dong, et al. (2001) Nature409:97-101). In contrast, PD-1 (programmed death 1) negatively regulatesT cell responses. PD-1 knockout mice develop lupus-like autoimmunedisease or autoimmune dilated cardiomyopathy (Nishimura, et al. (1999)Immunity 11:141-151; Nishimura, et al. (2001) Science 291:319-322). Theautoimmunity most likely results from the loss of signaling by bothligands PD-L1 and PD-L2. Recently, CD80 was identified as a secondreceptor for PD-L1 that transduces inhibitory signals into T cells(Butte, et al. (2007) Immunity 27:111-122).

The two inhibitory B7 family ligands, PD-L1 and PD-L2, have distinctexpression patterns. PD-L2 is inducibly expressed on DCs andmacrophages, whereas PD-L1 is broadly expressed on both hematopoieticcells and nonhematopoietic cell types (Okazaki & Honjo (2006) Immunology27:195-201; Keir, et al. (2008) Annu. Rev. Immunol. 26:677-704).Consistent with the immune-suppressive role of PD-1 receptor, a studyusing PD-L1^(−/−) and PD-L2^(−/−) mice has shown that both ligands haveoverlapping roles in inhibiting T cell proliferation and cytokineproduction (Keir, et al. (2006) J. Exp. Med. 203:883-895). PD-L1deficiency enhances disease progression in both the non-obese diabeticmodel of autoimmune diabetes and the mouse model of multiple sclerosis(experimental autoimmune encephalomyelitis (EAE); Anasari, et al. (2003)J. Exp. Med. 198:63-69; Salama, et al. (2003) J. Exp. Med. 198:71-78;Latchman, et al. (2004) Proc. Natl. Acad. Sci. USA. 101:10691-10696).PD-L1^(−/−) T cells produce elevated levels of the proinflammatorycytokines in both disease models. In addition, bone marrow chimeraexperiments have demonstrated that the tissue expression of PD-L1 (i.e.,within pancreas) uniquely contributes to its capacity of regionallycontrolling inflammation (Keir, et al. (2006) supra; Keir, et al. (2007)J. Immunol. 179:5064-5070; Grabie, et al. (2007) Circulation.116:2062-2071). PD-L1 is also highly expressed on placentalsyncytiotrophoblasts, which critically control the maternal immuneresponses to allogeneic fetus (Guleria, et al. (2005) J. Exp. Med.202:231-237).

Consistent with its immune-suppressive role, PD-L1 potently suppressesantitumor immune responses and helps tumors evade immune surveillance.PD-L1 can induce apoptosis of infiltrating cytotoxic CD8⁺ T cells, whichexpress a high level of PD-1 (Dong, et al. (2002) Nature 409:97-101;Dong & Chen (2003) J. Mol. Med. 81:281-287). Studies have shown thatblocking the PD-L1-PD-1 signaling pathway, in conjunction with otherimmune therapies, prevents tumor progression by enhancing antitumorcytotoxic T lymphocyte activity and cytokine production (Iwai, et al.(2002) Proc. Natl. Acad. Sci. USA 99:12293-12297; Blank, et al. (2004)Cancer Res. 64:1140-1145; Blank, et al. (2005) Cancer Immunol.Immunother. 54:307-314; Geng, et al. (2006) Int. J. Cancer.118:2657-2664). In addition, it has been shown that PD-L1 expression ondendritic cells promotes the induction of adaptive Foxp3⁺CD4⁺ regulatoryT cells (aT_(reg) cells), and PD-L1 is a potent inducer of aT_(reg)cells within the tumor microenvironment (Wang, et al. (2008) Proc. Natl.Acad. Sci. USA. 105:9331-9336).

An additional immune regulatory ligand, referred to as V-domain Igsuppressor of T cell activation (VISTA) or PD-L3, has been recentlyidentified as an upregulated molecule in a T cell transcriptionalprofiling screen. (Wang, et al. (2011) J. Exp. Med. 208:577; WO2011/120013). It has been shown that the extracellular Ig domain ofVISTA shares significant sequence homology with the B7 family ligandsPD-L1 and PD-L2, albeit with unique structural features that distinguishit from the B7 family members.

VISTA is primarily expressed on hematopoietic cells, and VISTAexpression is highly regulated on myeloid antigen-presenting cells(APCs) and T cells. Expression of VISTA on antigen presenting cells(APCs) suppresses T cell responses by engaging its counter-receptor on Tcells during cognate interactions between T cells and APCs. VISTAblockade enhances T cell-mediated immunity in an autoimmune diseasemodel, suggesting its unique and non-redundant role in controllingautoimmunity when compared with other inhibitory B7 family ligands suchas PD-L1 and PD-L2. In addition, VISTA blockade enhances anti-tumorimmunity and suppressed tumor growth in preclinical murine tumor models(WO 2011/120013). In this regard, therapeutic intervention of the VISTAinhibitory pathway represents a novel approach to modulate Tcell-mediated immunity for treating diseases such as viral infection andcancer.

SUMMARY OF THE INVENTION

The present invention provides an isolated VISTA antagonist thatcomprises a peptide that is identical to the amino acid sequence of SEQID NO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or amultimer, conjugate, analog, derivative or mimetic thereof.

In one embodiment, the isolated VISTA antagonist comprises a peptidewhich is identical to the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which comprises apeptide having an amino acid sequence that differs from SEQ ID NO:1 byat most 2 amino acid residues, or an multimer, conjugate, analog,derivative or mimetic thereof. In another embodiment, the isolated VISTAantagonist comprises a peptide having an amino acid sequence thatdiffers from SEQ ID NO:1 by at most 1 amino acid residue, or anmultimer, conjugate, analog, derivative or mimetic thereof. In yetanother embodiment, the isolated VISTA antagonist comprises a peptidewhich is identical to the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer, orconjugate thereof. In a specific embodiment, the isolated VISTAantagonist consists of the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His).

In one embodiment, the cysteine residues at positions 4 and 11 of SEQ IDNO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or theircorresponding positions in a variant of said peptide, form a disulfidebridge.

In another embodiment, the isolated VISTA antagonist has been modifiedto improve binding affinity and/or stability. In a specific embodiment,the isolated VISTA antagonist has been modified by PEG, acetylation,XTEN, albumin and/or multimerization.

In another embodiment, the isolated VISTA antagonist is directly orindirectly attached to an immunoglobulin polypeptide or a fragmentthereof. The immunoglobulin polypeptide may comprise a human IgG1, IgG2,IgG3 or IgG4 constant region or fragment thereof. Preferably, theimmunoglobulin polypeptide comprises a human IgG1 constant region orfragment thereof.

In yet another embodiment, the isolated VISTA antagonist comprisesmultiple, i.e., 2, 3, 4, 5, 6, 7 or more, copies of said peptide.

In a further embodiment, the isolated VISTA antagonist comprises anothermoiety that targets said peptide to a target site. The targeting moietymay be selected from an antibody or ligand that binds to an antigen, areceptor expressed by the target cell or an infectious agent.

In yet a further embodiment, the isolated VISTA antagonist is attachedto another moiety or another copy of said antagonist via a linker. Thelinker may be a peptide that permits the antagonist to interact withVISTA expressed on the surface of a target cell.

In a further embodiment, the isolated VISTA antagonist is directly orindirectly attached to a detectable label or therapeutic agent.

In several of the embodiments, the isolated VISTA antagonist binds tothe extracellular domain of VISTA and disrupts its interaction with aVISTA receptor and/or reduces or inhibits VISTA-mediated T cellsuppression.

In one embodiment, the isolated VISTA antagonist elicits anti-tumorand/or anti-viral activity.

Additionally, the invention contemplates a composition suitable fortherapeutic, prophylactic or diagnostic use comprising atherapeutically, prophylactically or diagnostically effective amount ofthe isolated VISTA antagonist.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier, diluent, solubilizer, preservative or mixturethereof.

In another embodiment, the composition further comprises anothertherapeutic agent, e.g., an anti-cancer agent, an anti-viral agent, acytokine or an immune agonist. In a particular embodiment, the othertherapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 or PD-L2fusion proteins, and EGFR antagonists.

In one embodiment, the composition is suitable for subcutaneousadministration or intravenous administration.

Moreover, the present invention further contemplates an isolated nucleicacid sequence encoding a VISTA antagonist peptide, analog, derivative ormimetic thereof disclosed herein, a vector containing the isolatednucleic acid sequence, and a host cell comprising the isolated nucleicacid sequence or the vector.

In one embodiment, the host cell is a mammalian cell, a bacterial cell,a fungal cell, a yeast cell, an avian cell or an insect cell.

The present invention further contemplates a method of expressing aVISTA antagonist peptide, analog, derivative or mimetic thereofcomprising culturing the host cell under conditions that provide forexpression of said peptide, analog, derivative or mimetic thereof.

Furthermore, the present invention contemplates various uses of theisolated VISTA antagonist.

In one embodiment, the invention provides a method for blocking,inhibiting or neutralizing VISTA-mediated T cell suppression, comprisingadministering to a subject in need thereof an effective amount of anisolated VISTA antagonist disclosed herein or a composition containingsaid isolated VISTA antagonist.

In another embodiment, the invention provides a method for stimulatingan immune response in a subject, comprising administering to the subjectin need thereof an effective amount of an isolated VISTA antagonistdisclosed herein or a composition containing said isolated VISTAantagonist. Such a method may be used for treating cancer in a subject.

The subject may have cancer and/or an infection selected from the groupconsisting of bacterial, viral, parasitic and fungal infections.

The bacterial infection may be caused by at least one bacterium selectedfrom the group consisting of Bordetella, Borrelia, Brucella,Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacterium,Enterobacter, Enterococcus, Erwinia, Escherichia, Francisella,Haemophilus, Heliobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pelobacter,Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus,Streptococcus, Treponema, Vibrio, Yersinia and Xanthomonas.

The viral infection may be caused by at least one virus selected fromthe group consisting of Adenoviridae, Papillomaviridae, Polyomaviridae,Herpesviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae,Caliciviridae, Picornaviridae, Coronoviridae, Flaviviridae,Retroviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae,Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae. Morespecifically, the virus may be adenovirus, herpes simplex type I, herpessimplex type 2, Varicella-zoster virus, Epstein-barr virus,cytomegalovirus, herpesvirus type 8, papillomavirus, BK virus, JC virus,smallpox, Hepatitis B, bocavirus, parvovirus B19, astrovirus, Norwalkvirus, coxsackievirus, Hepatitis A, poliovirus, rhinovirus, severe acuterespiratory syndrome virus, Hepatitis C, yellow fever, dengue virus,West Nile virus, rubella, Hepatitis E, human immunodeficiency virus(HIV), influenza, guanarito virus, Junin virus, Lassa virus, Machupovirus, Sabia virus, Crimean-Congo hemorrhagic fever virus, ebola virus,Marburg virus, measles virus, mumps virus, parainfluenza, respiratorysyncytial virus, human metapneumovirus, Hendra virus, Nipah virus,rabies, Hepatitis D, rotavirus, orbivirus, coltivirus or Banna virus.

The fungal infection may be selected from the group consisting ofthrush, candidiasis, cryptococcosis, histoplasmosis, blastomycosis,aspergillosis, coccidioidomycosis, paracoccidiomycosis, sporotrichosis,zygomycosis, chromoblastomycosis, lobomycosis, mycetoma, onychomycosis,piedra pityriasis versicolor, tinea barbae, tinea capitis, tineacorporis, tinea cruris, tinea favosa, tinea nigra, tinea pedis,otomycosis, phaeohyphomycosis, or rhinosporidiosis.

The parasitic infection may be caused by at least one parasite selectedfrom the group consisting of Entamoeba hystolytica, Giardia lamblia,Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatidarhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmaniabraziliensis, Leishmania tropica, Leishmania donovani, Toxoplasmagondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,Plasmodium falciparum, Trichomonas vaginalis, Histomonas meleagridis;Secementea; Trichuris trichiura, Ascaris lumbricoides, Enterobiusvermicularis, Ancylostoma duodenale, Necator americanus, Strongyloidesstercoralis, Wuchereria bancrofti, Dracunculus medinensis; blood flukes,liver flukes, intestinal flukes, lung flukes; Schistosoma mansoni,Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica,Fasciola gigantica, Heterophyes heterophyes, and Paragonimus westermani.

In another embodiment, the invention provides a method for enhancinganti-cancer or anti-tumor immunity, comprising administering to asubject in need thereof an effective amount of an isolated VISTAantagonist disclosed herein or a composition containing said isolatedVISTA antagonist.

In another embodiment, the invention provides a method for treating orpreventing cancer, inhibiting tumor invasion and/or cancer metastasis,comprising administering to a subject in need thereof an effectiveamount of an isolated VISTA antagonist disclosed herein or a compositioncontaining said isolated VISTA antagonist.

The cancer may be selected from the group consisting of carcinoma,lymphoma, blastoma, sarcoma, leukemia, lymphoid malignancies, melanoma,squamous cell cancer, lung cancer (including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer (including gastrointestinal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, head and neck cancer, B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; post-transplant lymphoproliferative disorder (PTLD), abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

In yet another embodiment, the invention provides a method for treatingor preventing a viral infection, comprising administering to a subjectin need thereof an effective amount of an isolated VISTA antagonistdisclosed herein or a composition containing said isolated VISTAantagonist.

These methods may further comprise the administration of anothertherapeutic agent, wherein said peptide and therapeutic may beseparately or jointly administered, at the same or different times.

In one embodiment, the other therapeutic agent is an anti-cancer agent,an anti-viral or other anti-infectious agent, a cytokine or an immuneagonist. Preferably, the other therapeutic agent is selected fromCTLA-4-Ig, anti-PD-1, PD-L1 or PD-L2 fusion proteins, and EGFRantagonists.

Finally, the present invention also contemplates a method for mappingthe active site of VISTA, comprising: (a) incubating an isolated VISTAfusion protein with an isolated VISTA antagonist comprising a peptidethat is identical to the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which comprises apeptide having an amino acid sequence which differs from SEQ ID NO:1 byat most 2 amino acid residues or an multimer, conjugate, analog,derivative or mimetic thereof; and (b) determining the binding site ofthe isolated VISTA antagonist.

In one embodiment, the active site of VISTA binds to a VISTA receptorand mediates immune suppression. In another embodiment, step (b)comprises domain deletion, domain swapping, amino acid mutagenesis, footprinting, NMR, X-ray crystallography or homology modeling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a VISTA antagonist peptide (SEQ ID NO:1) significantlyenhances the proliferation of T cells as compared to an anti-VISTAantibody (aVISTA) and an anti-PD-L1 antibody (aPDL1). Myeloid CD11B+APCs were incubated with OT2 CD4+ T cells, antigen, and a monoclonalantibody (aVISTA or aPDL1) or AP1049. Proliferation of T cells wasmeasured by tritium incorporation at 72 hours.

FIG. 2 shows that a VISTA antagonist peptide (SEQ ID NO:1) significantlyenhances anti-tumor immunity. Female mice inoculated with MB49 tumorswere treated with either PBS (control) or AP1049. Tumor size wasmeasured by caliper every 2-3 days.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein may be used inthe invention or testing of the present invention, suitable methods andmaterials are described herein. The materials, methods and examples areillustrative only, and are not intended to be limiting.

Definitions

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

“Antagonist,” as used herein, refers to a compound (preferably apeptide) that opposed the physiological effects of another compound. Forexample, at the receptor level, an antagonist is a compound that opposesthe receptor-associated response normally induced by another agent thatbinds to and activates the biological activity the receptor. Likewise,at the ligand level, an antagonist is a compound that opposes theligand-associated response normally induced when the ligand binds to itstarget receptor and/or accessory factors. In a specific embodiment, aVISTA antagonist is a compound, e.g., a peptide or analog, derivative ormimetic thereof, that binds to VISTA and opposes one or more of itsbiological activities, e.g., VISTA-mediated T cell suppression and/orVISTA-mediated suppression of anti-tumor immunity, thereby enhancing Tcell-mediated immunity and/or anti-tumor immunity.

“Analog,” as used herein, refers to a compound (preferably a peptide)whose structure is related to that of a given compound (preferably apeptide) but differs in chemical and biological properties.

“Antigen presenting cell,” as used herein, refers broadly toprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, and Langerhans cells) as well as other antigenpresenting cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, and oligodendrocytes).

“Amino acid,” as used herein refers broadly to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified(e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.) Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid (i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group), and an R group(e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.) Analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Allergic disease,” as used herein, refers broadly to a diseaseinvolving allergic reactions. More specifically, an “allergic disease”is defined as a disease for which an allergen is identified, where thereis a strong correlation between exposure to that allergen and the onsetof pathological change, and where that pathological change has beenproven to have an immunological mechanism. Herein, an immunologicalmechanism means that leukocytes show an immune response to allergenstimulation.

“Autoimmune disease” as used herein, refers broadly to a disease ordisorder arising from and directed against an individual's own tissuesor a co-segregate or manifestation thereof or resulting conditiontherefrom.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.)

“Conservatively modified variants,” as used herein, applies to bothamino acid and nucleic acid sequences, and with respect to particularnucleic acid sequences, refers broadly to conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein.“Silent variations” are one species of conservatively modified nucleicacid variations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) may be modifiedto yield a functionally identical molecule.

“Costimulatory receptor,” as used herein, refers broadly to receptorswhich transmit a costimulatory signal to an immune cell, e.g., CD28 orICOS.

“Cytoplasmic domain,” as used herein, refers broadly to the portion of aprotein which extends into the cytoplasm of a cell.

“Derivative” or “peptide derivative,” as used herein, contain amodification of one or more amino acid residues or a linker group orother covalently linked group. Non-limiting examples of derivativesinclude N-acyl derivatives of the amino terminal or of another freeamino group, esters of the carboxyl terminal or of another free carboxylor hydroxy group, amides of the carboxyl terminal or of another freecarboxyl group produced by reaction with ammonia or with a suitableamine, glycosylated derivatives, hydroxylated derivatives,nucleotidylated derivatives, ADP-ribosylated derivatives, pegylatedderivatives, phosphorylated derivatives, derivatives conjugated tolipophilic moieties, and derivatives conjugated to an antibody or otherbiological ligand. Also included among the chemical derivatives arethose obtained by modification of the peptide bond —CO—NH—, for exampleby reduction to —CH₂—NH— or alkylation to —CO—N(alkyl)-. Preferredderivatisation include, but are not limited tom C-terminal amidation andN-terminal acetylation, which removes the negative charge of the Cterminus or removes the positive charge at the N-terminus, respectively.Blocking of the C- or N-terminus, such as by C-terminal amidation orN-terminal acetylation, may improve proteolytic stability due to reducedsusceptibility to exoproteolytic digestion. Peptide derivatives having aC-terminal amide are represented with “NH₂” at the C-terminus.

“Diagnostic,” as used herein, refers broadly to identifying the presenceor nature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

“Diagnosing,” as used herein refers broadly to classifying a disease ora symptom, determining a severity of the disease, monitoring diseaseprogression, forecasting an outcome of a disease and/or prospects ofrecovery. The term “detecting” may also optionally encompass any of theforegoing. Diagnosis of a disease according to the present inventionmay, in some embodiments, be affected by determining a level of apolynucleotide or a polypeptide of the present invention in a biologicalsample obtained from the subject, wherein the level determined can becorrelated with predisposition to, or presence or absence of thedisease. It should be noted that a “biological sample obtained from thesubject” may also optionally comprise a sample that has not beenphysically removed from the subject.

“Effective amount,” as used herein, refers broadly to the amount of acompound, antibody, antigen, or cells that, when administered to apatient for treating a disease, is sufficient to effect such treatmentfor the disease. The effective amount may be an amount effective forprophylaxis, and/or an amount effective for prevention. The effectiveamount may be an amount effective to reduce, an amount effective toprevent the incidence of signs/symptoms, to reduce the severity of theincidence of signs/symptoms, to eliminate the incidence ofsigns/symptoms, to slow the development of the incidence ofsigns/symptoms, to prevent the development of the incidence ofsigns/symptoms, and/or effect prophylaxis of the incidence ofsigns/symptoms. The “effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, susceptibility,and pre-existing conditions, of the patient to be treated. The term“effective amount” is synonymous with “therapeutically effective amount”for purposes of this invention.

“Extracellular domain,” as used herein refers broadly to the portion ofa protein that extend from the surface of a cell.

“Expression vector,” as used herein, refers broadly to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including prokaryotic, yeast, fungal, plant, insect ormammalian cell. The term includes linear or circular expression systems.The term includes expression systems that remain episomal or integrateinto the host cell genome. The expression systems can have the abilityto self-replicate or not, i.e., drive only transient expression in acell. The term includes recombinant expression cassettes which containonly the minimum elements needed for transcription of the recombinantnucleic acid.

“Homology,” as used herein, refers broadly to a degree of similaritybetween a nucleic acid sequence and a reference nucleic acid sequence orbetween a polypeptide sequence and a reference polypeptide sequence.Homology may be partial or complete. Complete homology indicates thatthe nucleic acid or amino acid sequences are identical. A partiallyhomologous nucleic acid or amino acid sequence is one that is notidentical to the reference nucleic acid or amino acid sequence. Thedegree of homology can be determined by sequence comparison. The term“sequence identity” may be used interchangeably with “homology.”

“Host cell,” as used herein, refers broadly to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. Host cells maybe prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast,insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa,HEK-293, e.g., cultured cells, explants, and cells in vivo. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It should be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, progeny may not, in fact,be identical to the parent cell, but are still included within the scopeof the term as used herein.

“Immune response,” as used herein, refers broadly to T cell-mediatedand/or B cell-mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Bcell responses (e.g., antibody production) T cell responses (e.g.,cytokine production, and cellular cytotoxicity) and activation ofcytokine responsive cells, e.g., macrophages. As used herein, the term“down modulation” with reference to the immune response includes adiminution in any one or more immune responses, while the term “upmodulation” with reference to the immune response includes an increasein any one or more immune responses. It will be understood that upmodulation of one type of immune response may lead to a correspondingdownmodulation in another type of immune response. For example, upmodulation of the production of certain cytokines (e.g., IL-10) can leadto downmodulation of cellular immune responses.

“Inflammatory disease,” as used herein, refers broadly to chronic oracute inflammatory diseases.

“Detectable label” as used herein, refers broadly to a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means.

“Mimetic” or “peptidomimetic,” as used herein, refers to a fully orpartially synthetic peptide that has the activity of a given peptide.Such a mimetic or peptidomimetic comprises one or more amino acidresidues that is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, naturally occurring amino acid polymersand non-naturally occurring amino acid polymers.

Modifications of the VISTA and VISTA conjugate polypeptides describedherein include, but are not limited to N-terminus modification,C-terminus modification, peptide bond modification (e.g., CH₂—NH, CH₂—S,CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH, CH═CH or CF═CH), backbonemodifications, and residue modification, e.g., by the addition ofcarbohydrate residues to form glycoproteins, by the addition of chemicalresidues such as PEG and/or XTEN, etc. Methods for preparingpeptidomimetic compounds are well known in the art. Martin, (2010).

“Nucleic acid” or “nucleic acid sequence,” as used herein, refersbroadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide ineither single- or double-stranded form. The term encompasses nucleicacids, i.e., oligonucleotides, containing known analogs of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The term nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Polypeptide,” “peptide” and “protein,” are used interchangeably andrefer broadly to a polymer of amino acid residues. The terms apply toamino acid polymers in which one or more amino acid residue is an analogor mimetic of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Polypeptides can be modified, e.g., by theaddition of carbohydrate residues to form glycoproteins. The terms“polypeptide,” “peptide” and “protein” include glycoproteins, as well asnon-glycoproteins.

“Prophylactically effective amount,” as used herein, refers broadly tothe amount of a compound that, when administered to a patient forprophylaxis of a disease or prevention of the reoccurrence of a disease,is sufficient to effect such prophylaxis for the disease orreoccurrence. The prophylactically effective amount may be an amounteffective to prevent the incidence of signs and/or symptoms. The“prophylactically effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, predisposition toconditions, preexisting conditions, of the patient to be treated.

“Prophylaxis,” as used herein, refers broadly to a course of therapywhere signs and/or symptoms are not present in the patient, are inremission, or were previously present in a patient. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient. Further, prevention includes treating patients who maypotentially develop the disease, especially patients who are susceptibleto the disease (e.g., members of a patent population, those with riskfactors, or at risk for developing the disease).

“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

“Sequence identity,” as used herein, refers broadly to a degree ofsimilarity between a nucleic acid sequence and a reference nucleic acidsequence or between a polypeptide sequence and a reference polypeptidesequence. Sequence identity (also synonymous with “homology”) may bepartial or complete. Complete sequence identity indicates that thenucleic acid or amino acid sequences are identical, i.e., 100% sequenceidentity. A partially homologous nucleic acid or amino acid sequence isone that is not identical to the reference nucleic acid or amino acidsequence. The degree of homology can be determined by sequencecomparison, e.g., 60% identity, 70% identity, 80% identity, 90%identity, 95% identity, 97% identity, 98% identity, or 99% identity.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Subject,” as used herein, refers broadly to any animal that is in needof treatment either to alleviate a disease state or to prevent theoccurrence or reoccurrence of a disease state. Also, “subject” as usedherein, refers broadly to any animal that has risk factors, a history ofdisease, susceptibility, symptoms, and signs, was previously diagnosed,is at risk for, or is a member of a patient population for a disease.The subject may be a clinical patient such as a human or a veterinarypatient such as a companion, domesticated, livestock, exotic, or zooanimal. The term “subject” may be used interchangeably with the term“patient.”

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“T cell,” as used herein, refers broadly to CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting, or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, treatment, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. Therapy encompasses an alleviation of signsand/or symptoms in patients with ongoing disease signs and/or symptoms(e.g., inflammation, pain). Therapy also encompasses “prophylaxis”. Theterm “reduced”, for purpose of therapy, refers broadly to the clinicalsignificant reduction in signs and/or symptoms. Therapy includestreating relapses or recurrent signs and/or symptoms (e.g.,inflammation, pain). Therapy encompasses but is not limited toprecluding the appearance of signs and/or symptoms anytime as well asreducing existing signs and/or symptoms and eliminating existing signsand/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., inflammation, pain).

“Transmembrane domain,” as used herein, refers broadly to an amino acidsequence of about 15 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an alpha-helical structure. In an embodiment, atleast 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, et al. (1996) Annu. Rev. Neurosci. 19:235-263.

“Tumor,” as used herein, refers broadly to at least one cell or cellmass in the form of a tissue neoformation, in particular in the form ofa spontaneous, autonomous and irreversible excess growth, which is moreor less disinhibited, of endogenous tissue, which growth is as a ruleassociated with the more or less pronounced loss of specific cell andtissue functions. This cell or cell mass is not effectively inhibited,in regard to its growth, by itself or by the regulatory mechanisms ofthe host organism, e.g., melanoma or carcinoma. Tumor antigens not onlyinclude antigens present in or on the malignant cells themselves, butalso include antigens present on the stromal supporting tissue of tumorsincluding endothelial cells and other blood vessel components.

“Vector,” as used herein, refers broadly to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Vectors are referred to herein as “recombinant expression vectors” orsimply “expression vectors”. In general, expression vectors of utilityin recombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. The techniques and procedures are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See, e.g., Sambrook,et al. (2001) Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring HarborLaboratory Press. Standard techniques may be used for recombinant DNA,oligonucleotide synthesis, and tissue culture, and transformation (e.g.,electroporation, lipofection). Enzymatic reactions and purificationtechniques may be performed according to manufacturer's specificationsor as commonly accomplished in the art or as described herein.

The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesmay be used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

VISTA and VISTA Antagonists

This application relates to a peptide antagonist that can recognize andsuppress the inhibitory activity of VISTA. This peptide, designatedherein as AP1049, was discovered through phage display and shown toexhibit superior bioactivity when compared to an anti-VISTA monoclonalantibody. Given its neutralizing activity, AP1049 can be used to, e.g.,treat cancer and/or pathogenic, i.e., bacterial, fungal, parasite orviral infections and enhance anti-tumor immune responses and suppresstumor growth.

Accordingly, the present invention is a VISTA antagonistic peptide, aswell as multimers, conjugates, analogs, derivatives and mimetics thereofand methods of using this peptide to inhibit or suppress the activity ofVISTA. As used herein, the term “peptide” denotes an amino acid polymerthat is composed of at least two amino acids covalently linked by anamide bond. Peptides of the present invention are desirably 10 to 20residues in length, or more desirably 12 to residues in length. Incertain embodiments, a VISTA antagonistic peptide is a 12 to 20 residuepeptide containing the amino acid sequence of SEQ ID NO:1. In otherembodiments of the present invention, the isolated VISTA antagonistcomprises a peptide that is identical to the amino acid sequence of SEQID NO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or whichcomprises a peptide having an amino acid sequence that differs from SEQID NO:1 by at most 1 amino acid residue or at most 2 amino acidresidues, or an multimer, conjugate, analog, derivative or mimeticthereof. In yet other embodiments of the invention, the isolated VISTAantagonist consists of the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His).

In certain embodiments of the present invention, cysteine residues atpositions 4 and 11 of the VISTA antagonistic peptide (or theircorresponding positions in a variant of the VISTA antagonist) form adisulfide bridge.

In accordance with the present invention, multimers, conjugates,analogs, derivatives and mimetics of the peptide of the invention arealso provided.

An analog is a molecule that differs in chemical structure from a parentcompound, for example a homolog (differing by an increment in thechemical structure, such as a difference one amino acid residue), astructure that differs by one or more functional groups, or a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28).

Analogs can be prepared by modifying the amino acids sequence of SEQ IDNO:1. The simplest modifications involve the substitution of one or moreamino acids for amino acids having similar physiochemical and/orstructural properties. These so-called conservative substitutions arelikely to have minimal impact on the activity and/or structure of theresultant peptide. Examples of conservative substitutions includesubstituting a serine with a threonine, substituting alanine with aserine or valine, substituting aspartic acid with glutamic acid,substituting tryptophan with a tyrosine, substituting isoleucine withleucine or valine, substituting arginine with lysine, and/orsubstituting histidine with arginine or lysine. Conservativesubstitutions generally maintain (a) the structure of the peptidebackbone in the area of the substitution, for example, as a helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

Amino acid substitutions are typically classified in one or morecategories, including polar, hydrophobic, acidic, basic and aromatic,according to their side chains. Examples of polar amino acids includethose having side chain functional groups such as hydroxyl, sulfhydryl,and amide, as well as the acidic and basic amino acids. Polar aminoacids include, without limitation, asparagine, cysteine, glutamine,histidine, selenocysteine, serine, threonine, tryptophan and tyrosine.Examples of hydrophobic or non-polar amino acids include those residueshaving non-polar aliphatic side chains, such as, without limitation,leucine, isoleucine, valine, glycine, alanine, proline, methionine andphenylalanine. Examples of basic amino acid residues include thosehaving a basic side chain, such as an amino or guanidino group. Basicamino acid residues include, without limitation, arginine, homolysineand lysine. Examples of acidic amino acid residues include those havingan acidic side chain functional group, such as a carboxy group. Acidicamino acid residues include, without limitation aspartic acid andglutamic acid. Aromatic amino acids include those having an aromaticside chain group. Examples of aromatic amino acids include, withoutlimitation, biphenylalanine, histidine, 2-napthylalananine,pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It isnoted that some amino acids are classified in more than one group, forexample, histidine, tryptophan and tyrosine are classified as both polarand aromatic amino acids. Additional amino acids that are classified ineach of the above groups are known to those of ordinary skill in theart.

As used herein, a peptide derivative is a molecule which retains theprimary amino acids of the peptide, however, the N-terminus, C-terminus,and/or one or more of the side chains of the amino acids therein havebeen chemically altered or derivatized. Such derivatized peptidesinclude, for example, naturally occurring amino acid derivatives, forexample, 4-hydroxyproline for proline, 5-hydroxylysine for lysine,homoserine for serine, ornithine for lysine, and the like. Otherderivatives or modifications include, e.g., a label, such as fluoresceinor tetramethylrhodamine; or one or more post-translational modificationssuch as acetylation, amidation, formylation, hydroxylation, methylation,phosphorylation, sulfatation, glycosylation, or lipidation. Indeed,certain chemical modifications, in particular N-terminal glycosylation,have been shown to increase the stability of peptides in human serum(Powell et al. (1993) Pharma. Res. 10:1268-1273). Peptide derivativesalso include those with increased membrane permeability obtained byN-myristoylation (Brand, et al. (1996) Am. J. Physiol. Cell. Physiol.270:C1362-C1369).

In addition, a peptide derivative of the invention can include acell-penetrating sequence which facilitates, enhances, or increases thetransmembrane transport or intracellular delivery of the peptide into acell. For example, a variety of proteins, including the HIV-1 Tattranscription factor, Drosophila Antennapedia transcription factor, aswell as the herpes simplex virus VP22 protein have been shown tofacilitate transport of proteins into the cell (Wadia and Dowdy (2002)Curr. Opin. Biotechnol. 13:52-56). Further, an arginine-rich peptide(Futaki (2002) Int. J. Pharm. 245:1-7), a polylysine peptide containingTat PTD (Hashida, et al. (2004) Br. J. Cancer 90(6):1252-8), Pep-1(Deshayes, et al. (2004) Biochemistry 43(6):1449-57) or an HSP70 proteinor fragment thereof (WO 00/31113) is suitable for enhancingintracellular delivery of a peptide or mimetic of the invention into thecell.

While a peptide of the invention can be derivatized with by one of theabove indicated modifications, it is understood that a peptide of thisinvention may contain more than one of the above described modificationswithin the same peptide.

A mimetic or peptidomimetic refers to a synthetic chemical compoundwhich has substantially the same structural and/or functionalcharacteristics of a peptide of the invention. The mimetic can beentirely composed of synthetic, non-natural amino acid analogues, or canbe a chimeric molecule including one or more natural peptide amino acidsand one or more non-natural amino acid analogs. The mimetic can alsoincorporate any number of natural amino acid conservative substitutionsas long as such substitutions do not destroy the activity of themimetic. Routine testing can be used to determine whether a mimetic hasthe requisite activity, e.g., that it can inhibit the activity of VISTA.

The phrase “substantially the same,” when used in reference to a mimeticor peptidomimetic, means that the mimetic or peptidomimetic has one ormore activities or functions of the referenced molecule, e.g., theability to enhance T cell proliferation.

There are clear advantages for using a mimetic of a given peptide. Forexample, there are considerable cost savings and improved patientcompliance associated with peptidomimetics, since they can beadministered orally compared with parenteral administration forpeptides. Furthermore, peptidomimetics are much cheaper to produce thanpeptides.

Thus, a peptide of this invention has utility in the development of suchsmall chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. The techniques ofdeveloping peptidomimetics are conventional. For example, peptide bondscan be replaced by non-peptide bonds or non-natural amino acids thatallow the peptidomimetic to adopt a similar structure, and thereforebiological activity, to the original peptide. Further modifications canalso be made by replacing chemical groups of the amino acids with otherchemical groups of similar structure. The development of peptidomimeticscan be aided by determining the tertiary structure of the originalpeptide by NMR spectroscopy, crystallography and/or computer-aidedmolecular modeling. These techniques aid in the development of novelcompositions of higher potency and/or greater bioavailability and/orgreater stability than the original peptide (Dean (1994) BioEssays16:683-687; Cohen & Shatzmiller (1993) J. Mol. Graph. 11:166-173; Wiley& Rich (1993) Med. Res. Rev. 13:327-384; Moore (1994) Trends Pharmacol.Sci. 15:124-129; Hruby (1993) Biopolymers 33:1073-1082; Bugg, et al.(1993) Sci. Am. 269:92-98). Once a potential peptidomimetic compound isidentified, it may be synthesized and assayed using an assay describedherein or any other appropriate assay for monitoring VISTA activity.

Peptide mimetic compositions can contain any combination of non-naturalstructural components, which are typically from three structural groups:residue linkage groups other than the natural amide bond (“peptidebond”) linkages; non-natural residues in place of naturally occurringamino acid residues; residues which induce secondary structural mimicry,i.e., induce or stabilize a secondary structure, e.g., a beta turn,gamma turn, beta sheet, alpha helix conformation, and the like; or otherchanges which confer resistance to proteolysis. For example, a peptidecan be characterized as a mimetic when one or more of the residues arejoined by chemical means other than an amide bond. Individualpeptidomimetic residues can be joined by amide bonds, non-natural andnon-amide chemical bonds other chemical bonds or coupling meansincluding, for example, glutaraldehyde, N-hydroxysuccinimide esters,bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropyl-carbodiimide (DIC). Linking groups alternative to theamide bond include, for example, ketomethylene (e.g., —C(═O)—CH₂— for—C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether(CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide,thioamide, or ester (see, e.g., Spatola (1983) in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, 7:267-357, “Peptideand Backbone Modifications,” Marcel Decker, NY).

As discussed, a peptide can be characterized as a mimetic by containingone or more non-natural residues in place of a naturally occurring aminoacid residue. Non-natural residues are known in the art. Particularnon-limiting examples of non-natural residues useful as mimetics ofnatural amino acid residues are mimetics of aromatic amino acidsinclude, for example, D- or L-naphylalanine; D- or L-phenylglycine; D-or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- orL-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- orL-p-biphenylphenylalanine; D- or L-p-methoxy-biphenyl-phenylalanine; andD- or L-2-indole(alkyl)alanines, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid. Aromaticrings of a non-natural amino acid that can be used in place a naturalaromatic ring include, for example, thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution withnon-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; and sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) including, for example,1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylgroups can also be converted to asparaginyl and glutaminyl groups byreaction with ammonium ions.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate.

One or more residues can also be replaced by an amino acid (orpeptidomimetic residue) of the opposite chirality. Thus, any amino acidnaturally occurring in the L-configuration (which can also be referredto as R or S, depending upon the structure of the chemical entity) canbe replaced with the same amino acid or a mimetic, but of the oppositechirality, referred to as the D-amino acid, but which can additionallybe referred to as the R- or S-form.

As will be appreciated by one skilled in the art, a peptidomimetic ofthe present invention can also include one or more of the modificationsdescribed herein for derivatized peptides, e.g., a detectable label(such as an effector label or a radionuclide), a therapeutic agent (suchas a chemotherapeutic agent), one or more post-translationalmodifications, or cell-penetrating sequence.

For example, the VISTA antagonists described herein may be modifiedpost-translationally to add effector labels such as chemical linkers,detectable labels such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent labels, or functional labels such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials. Further exemplary enzymes include, but are notlimited to, horseradish peroxidase, acetylcholinesterase, alkalinephosphatase, β-galactosidase and luciferase. Further exemplaryfluorescent materials include, but are not limited to, rhodamine,fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin and dansyl chloride. Furtherexemplary chemiluminescent labels include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin, luciferase, and aequorin. Further exemplaryradioactive materials include, but are not limited to, bismuth-213(²¹³Bs), carbon-14 (¹⁴C), carbon-11 (¹¹C), chlorine-C18 (Cl¹⁸),chromium-51 (⁵¹Cr), cobalt-57 (⁵⁷Co), cobalt-60 (⁶⁰Co), copper-64(⁶⁴Cu), copper-67 (⁶⁷Cu), dysprosium-165 (¹⁶⁵Dy), erbium-169 (¹⁶⁹Er),fluorine-18 (¹⁸F), gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), germanium-68(⁶⁸Ge), holmium-166 (¹⁶⁶Ho), indium-111 (¹¹¹In), iodine-125 (¹²⁵I),iodine-123 (¹²⁴I), iodine-124 (¹²⁴I), iodine-131 (¹³¹I), iridium-192(¹⁹²Ir), iron-59 (⁵⁹Fe), krypton-81 (⁸¹Kr), lead-212 (²¹²Pb),lutetium-177 (¹⁷⁷Lu), molybdenum-99 (⁹⁹Mo), nitrogen-13 (¹³N), oxygen-15(¹⁵O), palladium-103 (¹⁰³Pd), phosphorus-32 (³²P), potassium-42 (⁴²K),rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re), rubidium-81 (⁸¹Rb),rubidium-82 (⁸²Rb), samarium-153 (¹⁵³Sm) , selenium-75 (⁷⁵Se), sodium-24(²⁴Na), strontium-82 (⁸²Sr), strontium-89 (⁸⁹Sr), sulfur 35 (³⁵S),technetium-99m (⁹⁹ Tc), thallium-201 (²⁰¹Tl), tritium (³H), xenon-133(¹³³Xe), ytterbium-169 (¹⁶⁹Yb), ytterbium-177 (¹⁷⁷Yb), and yttrium-90(⁹⁰Y).

Additionally, the VISTA antagonists provided herein may be modified toadd a therapeutic agent including, but not limited to, chemotherapeuticagents such as carboplatin, cisplatin, paclitaxel, gemcitabine,calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D,cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFRantagonists, platins, taxols, irinotecan, 5-fluorouracil, gemcytabine,leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids(e.g., vinblastine, vincristine, vindesine and vinorelbine), mustines,tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists,selective androgen receptor modulators, selective estrogen receptormodulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,interleukins (e.g., IL-12 or IL-2), IL-12R antagonists, Toxin conjugatedmonoclonal antibodies, tumor antigen specific monoclonal antibodies,Erbitux®, Avastin®, Pertuzumab, anti-CD20 antibodies, Rituxan®,ocrelizumab, ofatumumab, DXL625, Herceptin®, or any combination thereof.Toxic enzymes from plants and bacteria such as ricin, diphtheria toxinand Pseudomonas toxin may be conjugated to the VISTA antagonists togenerate cell-type-specific-killing reagents. Youle, et al. (1980) Proc.Nat'l Acad. Sci. USA 77:5483; Gilliland, et al. (1980) Proc. Nat'l Acad.Sci. USA 77:4539; Krolick, et al. (1980) Proc. Nat'l Acad. Sci. USA77:5419.

Furthermore, the VISTA antagonists described herein may be conjugated toa radionuclide that emits alpha or beta particles (e.g.,radioimmunoconjuagtes). Such radioactive isotopes include but are notlimited to beta-emitters such as phosphorus-32 (³²P), scandium-47(⁴⁷Sc), copper-67 (⁶⁷Cu), gallium-67 (⁶⁷Ga), yttrium-88 (^(H)Y),yttrium-90 (⁹⁰Y), iodine-125 (¹²⁵I), iodine-131 (¹³¹I), samarium-153(¹⁵³Sm), lutetium-177 (¹⁷⁷Lu), rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re),and alpha-emitters such as astatine-211 (²¹¹At), lead-212 (²¹²Pb),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi) or actinium-225 (²²⁵Ac).

Methods are known in the art for conjugating a VISTA antagonistdescribed herein to a label, such as those methods described by Hunter,et al (1962) Nature 144: 945; David, et al. (1974) Biochemistry 13:1014; Pain, et al. (1981) J. Immunol. Meth. 40: 219; and Nygren (1982)Histochem and Cytochem, 30: 407.

Additionally, the VISTA antagonists described herein may compriseanother moiety, i.e., a “targeting moiety,” that targets the antagonistpeptide to a target site (such as a cancer cell, a tumor, avirally-infected cell, etc). The targeting moiety may be selected froman antibody or ligand that binds to an antigen, a receptor expressed bythe target cell or an infectious agent.

The VISTA antagonist (as well as multimers, conjugates, analogs,derivatives and mimetics thereof) may also be directly or indirectlyattached to an immunoglobulin polypeptide or a fragment thereof, e.g., aantibody constant region.

A “conjugate,” as used herein, refers to a compound having at least oneisolated VISTA antagonist peptide and one immunoglobulin polypeptide ora fragment thereof, e.g., antibody constant region, joined at thepolypeptide level, with or without the use of a linker. A conjugate maybe a fusion polypeptide produced as the result of joining at the nucleicacid level of genes encoding at least one natriuretic peptide and oneantibody constant region, with or without a coding sequence for apeptide linker.

Such VISTA antagonist peptide-antibody conjugates may have a higherserum stability, e.g., at least 20%, preferably at least 30%, 50%, 80%,100%, 200% or more, increase in the serum half-life when compared withthe antagonist peptide without the antibody constant region under thesame conditions. A human antibody, e.g., a human IgG, such as IgG1,IgG2, IgG3 or IgG4, is frequently used to derive a constant region or afragment thereof for the purpose of making a natriuretic peptideconjugate of this invention.

As used herein, an “antibody (or immunoglobulin) constant region” refersto a polypeptide that corresponds to at least a portion of the constantregion of an antibody heavy chain or light chain, such portion includingat least one constant domain (e.g., the constant domain of CL or one ofthe constant domains of C_(H)). For example, an “antibody constantregion” used for making the conjugates of this invention may be derivedfrom an antibody heavy chain and include two out of three (C_(H)2 andC_(H)3 for IgA, IgD, and IgG) or three out of four (C_(H)2, CH3, andCH4, for IgE and IgM) constant domains; the first constant domain(C_(H)I) may be present in some cases but may be excluded in others.Such an antibody constant region can be obtained by a variety of means,e.g., by a recombinant method or synthetic method, or by purificationsubsequent to enzymatic digestion, for instance, pepsin or papaindigestion of an intact antibody or an antibody heavy or light chain.

Further encompassed by this term as used in this application arepolypeptides having a substantial sequence identity (for instance, atleast 80%, 85%, 90%, 95% or more) to the corresponding amino acidsequence of an antibody heavy or light chain constant region or aportion thereof that contains at least one constant domain nearest tothe C-terminus of the antibody chain, so long as the presence of such an“antibody constant region” in a VISTA antagonist peptide-antibodyconstant region conjugate renders the conjugate a higher serumstability.

Additionally, the peptide, multimer, conjugate, analog, derivative ormimetic may be modified to increase certain properties, e.g., biologicalhalf life. Various approaches are possible including, but not limitedto, N-terminal modification/conjugation (e.g., lipidation oracetylation), C-terminal modification/conjugation (e.g., lipidation oracetylation), amino acid substitutions (i.e., substitution of naturalamino acid with unnatural amino acids, such as D-conformation,N-methylation, tetra-substitution, beta-amino acids, etc.), peptidebackbone modifications (e.g., chemical modification of peptide bonds,such as simple reductions or replacement of carbonyl or amide groupswith esters, sulfides and alkyls), side chain modifications and/orcyclization (e.g., disulfide bond formation).

In one embodiment, the peptide may be pegylated to, e.g., increase thebiological (e.g., serum) half life of the antibody. To pegylate apeptide, typically it is reacted with polyethylene glycol (PEG), such asa reactive ester or aldehyde derivative of PEG, under conditions inwhich one or more PEG groups become attached to the peptide. Preferably,the pegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer.

Similarly, in another embodiment, a peptide, multimer, conjugate,analog, derivative or mimetic may be modified by conjugation ofpolysialic acid (PSA) to increase half-life.

Additionally, the peptide, multimer, conjugate, analog, derivative ormimetic may be modified, e.g., genetically fused or chemicallyconjugated, to comprise extended recombinant polypeptide (XTEN), througha process called XTENylation, to improve its half life. XTEN is a long,hydrophilic, and unstructured protein-based polymer of 864 amino acids.See, e.g., WO 2013/130683 which is herein expressly incorporated byreference in its entirety. When attached to a molecule of interest,greatly increases the effective size of the molecule, thereby prolongingits presence in serum by slowing kidney clearance in a manner analogousto that of PEG. In addition to slowing kidney clearance, attachment toXTEN can also inhibit receptor-mediated clearance by reducing theligand's affinity for its receptor. XTEN coupling chemistries include,but are not limited to, Thiol-XTEN; Maleimide-XTEN; Alkyne-XTEN; andIodoacetyl-XTEN.

Moreover, the peptide, multimer, conjugate, analog, derivative ormimetic may be modified with recombinant albumin, e.g., NovozymesRecombumin®, to improve half life. The peptide can be genetically fusedor chemically conjugated to a recombinant albumin using standardprotocols.

Furthermore, the peptide, multimer, conjugate, analog, derivative ormimetic may be modified by the addition and/or removal of specific aminoacids to and/or from the peptide. For example, a number of specificamino acids may be added to the peptide, thereby strengthening ortightening its molecular structure to make it less susceptible tobiological degradation and, thus, providing a longer life-span in theblood, using, e.g., Zealand Structure Induced Probe (SIP®) tailtechnology.

Yet another exemplary method for improving the stability and therapeuticpotential of peptides, analogs, derivatives or mimetics ismultimerization. For example, a multimer may comprise two or morecopies, e.g., 2, 3, 4, 5, 6, or more, of the isolated VISTA antagonistor variant thereof. Multimers include both homomultimers andheteromultimers. Multimerization can result in increased peptidestability, higher binding strength (due to multiple valencies in themolecule), and/or improved pharmacokinetic properties.

Another exemplary approach for improving the stability and, thus,therapeutic potential of the VISTA antagonist peptides, multimers,conjugates, analogs, derivatives or mimetics disclosed herein is theaddition of acetyl groups to the N and/or C terminus of the peptide.Acetylation may protect the peptide from exopeptidases, therebyextending the half-life of the peptide.

Production of VISTA Antagonists

The peptide multimer, conjugate, analog, derivative or mimetic can beproduced and isolated using any method known in the art. Peptides can besynthesized, whole or in part, using chemical methods known in the art(see, e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn(1980) Nucleic Acids Res. Symp. Ser. 225-232; and Banga (1995)Therapeutic Peptides and Proteins, Formulation, Processing and DeliverySystems, Technomic Publishing Co., Lancaster, Pa.). Peptide synthesiscan be performed using various solid-phase techniques (see, e.g.,Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol.289:3-13) and automated synthesis may be achieved, e.g., using the ABI431A Peptide Synthesizer (Perkin Elmer) in accordance with themanufacturer's instructions.

Individual synthetic residues and peptides incorporating mimetics can besynthesized using a variety of procedures and methodologies known in theart (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al.(Eds) John Wiley & Sons, Inc., NY). Peptides and peptide mimetics canalso be synthesized using combinatorial methodologies. Techniques forgenerating peptide and peptidomimetic libraries are well-known, andinclude, for example, multipin, tea bag, and split-couple-mix techniques(see, for example, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; and Ostresh (1996) Methods Enzymol. 267:220-234). Modifiedpeptides can be further produced by chemical modification methods (see,for example, Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel(1995) Free Radic. Biol. Med. 19:373-380; and Blommers (1994)Biochemistry 33:7886-7896).

Alternatively, a peptide of this invention can be prepared inrecombinant protein systems using polynucleotide sequences encoding thepeptides. By way of illustration, a nucleic acid molecule encoding apeptide of the invention is introduced into a host cell, such asbacteria, yeast or mammalian cell, under conditions suitable forexpression of the peptide, and the peptide is purified or isolated usingmethods known in the art. See, e.g., Deutscher et al. (1990) Guide toProtein Purification: Methods in Enzymology Vol. 182, Academic Press. Inparticular embodiments, the peptide, or analog, derivative or mimeticthereof is isolated and/or purified to homogeneity (e.g. greater than90% purity).

It is contemplated that the peptide disclosed herein can be used as alead compound for the design and synthesis of compounds with improvedefficacy, clearance, half-lives, and the like.

One approach includes structure-activity relationship (SAR) analysis(e.g., NMR analysis) to determine specific binding interactions betweenthe peptide and VISTA to facilitate the development of more efficaciousagents. Agents identified in such SAR analysis or from agent librariescan then be screened for their ability to, e.g., decrease the activityof VISTA and/or enhance T cell proliferation.

Pharmaceutical Compositions

The VISTA antagonist peptide, multimer, conjugate, analog, derivativeand mimetic thereof described herein can be provided in a pharmaceuticalcomposition.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, epicutaneous,epidural, inhalation, intraarterial, intracardial,intracerebroventricular, intradermal, intramuscular, intranasal,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration may occur by means of injection, powder, liquid, gel,drops, or other means of administration.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations may be found, for example, in Grennaro (2005)[Ed.] Remington: The Science and Practice of Pharmacy [21^(st) Ed.]

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition may be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriermay be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition.

The VISTA antagonist peptide, multimer, conjugate, analog, derivativeand mimetic thereof described herein may be formulated intopharmaceutical compositions of various dosage forms. To prepare thepharmaceutical compositions of the invention, at least one VISTAantagonist as the active ingredient may be intimately mixed withappropriate carriers and additives according to techniques well known tothose skilled in the art of pharmaceutical formulations. See Grennaro(2005) [Ed.] Remington: The Science and Practice of Pharmacy [21^(st)Ed.] For example, the antagonists described herein may be formulated inphosphate buffered saline pH 7.2 and supplied as a 5.0 mg/mL clearcolorless liquid solution.

Similarly, compositions for liquid preparations include solutions,emulsions, dispersions, suspensions, syrups, and elixirs, with suitablecarriers and additives including but not limited to water, alcohols,oils, glycols, preservatives, flavoring agents, coloring agents, andsuspending agents. Typical preparations for parenteral administrationcomprise the active ingredient with a carrier such as sterile water orparenterally acceptable oil including but not limited to polyethyleneglycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, withother additives for aiding solubility or preservation may also beincluded. In the case of a solution, it may be lyophilized to a powderand then reconstituted immediately prior to use. For dispersions andsuspensions, appropriate carriers and additives include aqueous gums,celluloses, silicates, or oils.

For each of the recited embodiments, the VISTA antagonist peptides,multimers, conjugates, analogs, derivatives and mimetics thereof may beadministered by a variety of dosage forms. Any biologically-acceptabledosage form known to persons of ordinary skill in the art, andcombinations thereof, are contemplated. Examples of such dosage formsinclude, without limitation, reconstitutable powders, elixirs, liquids,solutions, suspensions, emulsions, powders, granules, particles,microparticles, dispersible granules, cachets, inhalants, aerosolinhalants, patches, particle inhalants, implants, depot implants,injectables (including subcutaneous, intramuscular, intravenous, andintradermal), infusions, and combinations thereof.

In many cases, it will be preferable to include isotonic agents, e.g.,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions maybe brought about by including in the composition an agent which delaysabsorption, e.g., monostearate salts and gelatin. Moreover, thecompounds described herein may be formulated in a time releaseformulation, e.g. in a composition that includes a slow release polymer.The VISTA and VISTA conjugate may be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers may be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations are knownto those skilled in the art.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings inGoodman, et al. (2011) Goodman & Gilman's The Pharmacological Basis ofTherapeutics [12^(th) Ed.]; Howland, et al. (2005) Lippincott'sIllustrated Reviews: Pharmacology [2^(nd) Ed.]; and Golan, (2008)Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy[2^(nd) Ed.] See, also, Grennaro (2005) [Ed.] Remington: The Science andPractice of Pharmacy [21^(st) Ed.]

The compositions described herein may be administered in any of thefollowing routes: buccal, epicutaneous, epidural, infusion, inhalation,intraarterial, intracardial, intracerebroventricular, intradermal,intramuscular, intranasal, intraocular, intraperitoneal, intraspinal,intrathecal, intravenous, oral, parenteral, pulmonary, rectally via anenema or suppository, subcutaneous, subdermal, sublingual, transdermal,and transmucosal. The preferred routes of administration are intravenousinjection or infusion. The administration can be local, where thecomposition is administered directly, close to, in the locality, near,at, about, or in the vicinity of, the site(s) of disease, e.g., tumor,or systemic, wherein the composition is given to the patient and passesthrough the body widely, thereby reaching the site(s) of disease. Localadministration (e.g., injection) may be accomplished by administrationto the cell, tissue, organ, and/or organ system, which encompassesand/or is affected by the disease, and/or where the disease signs and/orsymptoms are active or are likely to occur (e.g., tumor site).Administration can be topical with a local effect, composition isapplied directly where its action is desired (e.g., tumor site).

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms as known in the art. Anybiologically-acceptable dosage form known to persons of ordinary skillin the art, and combinations thereof, are contemplated. Examples of suchdosage forms include, without limitation, chewable tablets, quickdissolve tablets, effervescent tablets, reconstitutable powders,elixirs, liquids, solutions, suspensions, emulsions, tablets,multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules,hard gelatin capsules, caplets, lozenges, chewable lozenges, beads,powders, gum, granules, particles, microparticles, dispersible granules,cachets, douches, suppositories, creams, topicals, inhalants, aerosolinhalants, patches, particle inhalants, implants, depot implants,ingestibles, injectables (including subcutaneous, intramuscular,intravenous, and intradermal), infusions, and combinations thereof.

Other compounds which can be included by admixture are, for example,medically inert ingredients (e.g., solid and liquid diluent), such aslactose, dextrosesaccharose, cellulose, starch or calcium phosphate fortablets or capsules, olive oil or ethyl oleate for soft capsules andwater or vegetable oil for suspensions or emulsions; lubricating agentssuch as silica, talc, stearic acid, magnesium or calcium stearate and/orpolyethylene glycols; gelling agents such as colloidal clays; thickeningagents such as gum tragacanth or sodium alginate, binding agents such asstarches, arabic gums, gelatin, methylcellulose, carboxymethylcelluloseor polyvinylpyrrolidone; disintegrating agents such as starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuff; sweeteners; wetting agents such as lecithin, polysorbates orlaurylsulphates; and other therapeutically acceptable accessoryingredients, such as humectants, preservatives, buffers andantioxidants, which are known additives for such formulations.

Liquid dispersions for oral administration can be syrups, emulsions,solutions, or suspensions. The syrups can contain as a carrier, forexample, saccharose or saccharose with glycerol and/or mannitol and/orsorbitol. The suspensions and the emulsions can contain a carrier, forexample a natural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol.

In further embodiments, the present invention provides kits includingone or more containers comprising pharmaceutical dosage units comprisingan effective amount of one or more VISTA antagonists of the presentinvention. Kits may include instructions, directions, labels, marketinginformation, warnings, or information pamphlets.

The amount of VISTA antagonist in a therapeutic composition according toany embodiments of this invention may vary according to factors such asthe disease state, age, gender, weight, patient history, risk factors,predisposition to disease, administration route, pre-existing treatmentregime (e.g., possible interactions with other medications), and weightof the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of therapeutic situation.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of antibodies, and fragmentsthereof, calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the antibodies, and fragmentsthereof, and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an antibodies, andfragments thereof, for the treatment of sensitivity in individuals. Intherapeutic use for treatment of conditions in mammals (e.g., humans)for which the antibodies and fragments thereof of the present inventionor an appropriate pharmaceutical composition thereof are effective, theantibodies and fragments thereof of the present invention may beadministered in an effective amount. The dosages as suitable for thisinvention may be a composition, a pharmaceutical composition or anyother compositions described herein.

The dosage may be administered as a single dose, a double dose, a tripledose, a quadruple dose, and/or a quintuple dose. The dosages may beadministered singularly, simultaneously, and sequentially.

The dosage form may be any form of release known to persons of ordinaryskill in the art. The compositions of the present invention may beformulated to provide immediate release of the active ingredient orsustained or controlled release of the active ingredient. In a sustainedrelease or controlled release preparation, release of the activeingredient may occur at a rate such that blood levels are maintainedwithin a therapeutic range but below toxic levels over an extendedperiod of time (e.g., 4 to 24 hours). The preferred dosage forms includeimmediate release, extended release, pulse release, variable release,controlled release, timed release, sustained release, delayed release,long acting, and combinations thereof, and are known in the art.

As defined herein, a therapeutically effective amount of VISTAantagonist peptide, analog, derivative or mimetic thereof (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a peptide can include a single treatment or,preferably, can include a series of treatments.

In a preferred example, a subject is treated with peptide, analog,derivative or mimetic thereof in the range of between about 0.1 to 20mg/kg body weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

It will be appreciated that the pharmacological activity of thecompositions may be monitored using standard pharmacological models thatare known in the art. Furthermore, it will be appreciated that thecompositions comprising a VISTA antagonist may be incorporated orencapsulated in a suitable polymer matrix or membrane for site-specificdelivery, or may be functionalized with specific targeting agentscapable of effecting site specific delivery. These techniques, as wellas other drug delivery techniques are well known in the art.Determination of optimal dosages for a particular situation is withinthe capabilities of those skilled in the art. See, e.g., Grennaro (2005)[Ed.] Remington: The Science and Practice of Pharmacy [21^(st) Ed.]

A peptide or analog, derivative or mimetic of this invention can beco-formulated with and/or coadministered with one or more additionaltherapeutic agents (e.g., an anti-cancer agent, an anti-viral agent, acytokine and/or an immune agonist). Such combination therapies mayrequire lower dosages of the peptide or analog, derivative or mimeticand/or the co-administered agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies. There are anumber of agents that may be advantageously combined with peptide oranalog, derivative or mimetic of the invention and the selection of suchagents will depend on the intended disease or condition to be treated.For example, the present invention includes combination therapiescomposed of a peptide or multimer, conjugate, analog, derivative ormimetic of the invention that is capable of inducing or promoting aresponse against a cancerous or pre-cancerous condition and at least oneanti-cancer agent. Accordingly, in particular embodiments, the instantpeptide or analog, derivative or mimetic is used as an adjuvant therapyin the treatment of cancer. As another example, the invention embracescombination therapies that include a peptide or analog, derivative ormimetic of the invention that is capable of inducing or promoting atherapeutic response against a viral infection and at least oneanti-viral agent. Exemplary therapeutic agents that may be contained inthe compositions comprising the VISTA antagonist peptide, multimer,conjugate, analog, derivative or mimetic include, e.g., CTLA-4-Ig,anti-PD-1, PD-L1 or PD-L2 fusion proteins and EGFR antagonists.

Anti-cancer agents include, but are not limited to, cytotoxic agentssuch as Vinca alkaloid, taxanes, and topoisomerase inhibitors; antisensenucleic acids such as augmerosen/G3139, LY900003 (ISIS 3521), ISIS 2503,OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome encapsulated c-rafantisense oligonucleotide/ISIS-5132), MG98, and other antisense nucleicacids that target PKCα, clusterin, IGFBPs, protein kinase A, cyclin D1,or Bcl-2; anticancer nucleozymes such as angiozyme (RibozymePharmaceuticals); tumor suppressor-encoding nucleic acids such as a p53,BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC; oncolytic virusessuch as oncolytic adenoviruses and herpes viruses; anti-cancerimmunogens such as a cancer antigen/tumor-associated antigen, e.g., anepithelial cell adhesion molecule (Ep-CAM/TACSTD1), mucin 1 (MUC1),carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72(TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associatedviral vaccines, tumor-derived heat shock proteins, and the like;anti-cancer cytokines, chemokines, or combination thereof; inhibitors ofangiogenesis, neovascularization, and/or other vascularization; and/orany other conventional anticancer agent including fluoropyrimidinercarbamates, non-polyglutamatable thymidylate synthase inhibitors,nucleoside analogs, antifolates, topoisomerase inhibitors, polyamineanalogs, mTOR inhibitors, alkylating agents, lectin inhibitors, vitaminD analogs, carbohydrate processing inhibitors, anti-metabolism folateantagonists, thumidylate synthase inhibitors, antimetabolites,ribonuclease reductase inhibitors, dioxolate nucleoside analogs, andchemically modified tetracyclines.

Anti-viral agents of use in the invention include, but are not limitedto, protease inhibitors (e.g., acyclovir) in the context of HIVtreatment or an anti-viral antibody (e.g., an anti-gp41 antibody in thecontext of HIV treatment; an anti-CD4 antibody in the context of thetreatment of CMV, etc.). Numerous other types of anti-viral agents areknown in the art.

Toxicity and therapeutic efficacy of the peptide or analog, derivativeor mimetic can be determined by standard pharmaceutical procedures incell cultures or experimental animals. The data obtained from the cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such agents lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any agent used in the methods of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Use of VISTA Antagonists and Compositions Comprising the Same

The peptide or analog, derivative or mimetic of this invention finds usein inhibiting the activity of VISTA (i.e., PD-L3) thereby upregulatingimmune responses. Upregulation of immune responses can be in the form ofenhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through inhibitionof VISTA activity is useful in the prevention and/or treatment ofinfections with microbes, e.g., bacteria, viruses, or parasites, or incases of immunosuppression and cancer.

Accordingly, the present invention includes prophylactic and therapeuticmethods for the prevention and treatment of cancer and infectiousdisease. Terms such as “treat,” “treating” and “treatment” herein referto the delivery of an effective amount of a peptide or analog,derivative or mimetic of this invention with the purpose of easing,ameliorating, or eradicating (curing) such symptoms or disease statesalready developed. The terms “prevent,” “preventing” and “prevention”refer to the delivery of an effective amount of a peptide or analog,derivative or mimetic of this invention with the purpose of preventingany symptoms or disease state to develop. Thus, these terms are meant toinclude prophylactic treatment.

Accordingly to one embodiment, the invention provides a method oftreating or preventing cancer, inhibiting tumor invasion and/or cancermetastasis by administering to a subject in need thereof, such as amammalian subject, preferably a human subject, an effective amount of anisolated VISTA antagonist disclosed herein or a composition containingsaid isolated VISTA antagonist. Optionally the subject has one or moreprecancerous lesions or is predisposed to cancer, e.g., as a result ofgenetic mutation, family history or exposure to a carcinogenic agent. Inanother embodiment the invention provides a method of treating cancer insubject, such as a mammalian subject, preferably a human subject, suchas a human subject, who optionally has a detectable level of cancercells. In accordance with these embodiments, the subject is administereda peptide or analog, derivative or mimetic of this invention in anamount sufficient to detectably reduce the development or progression ofthe cancer in the subject.

Cancers are generally composed of single or several clones of cells thatare capable of partially independent growth in a host (e.g., a benigntumor) or fully independent growth in a host (malignant cancer). Cancercells are cells that divide and reproduce abnormally with uncontrolledgrowth.

Cancer cells arise from host cells via neoplastic transformation (i.e.,carcinogenesis). Terms such as “preneoplastic,” “premalignant” and“precancerous” with respect to the description of cells and/or tissuesherein refer to cells or tissues having a genetic and/or phenotypicprofile that signifies a significant potential of becoming cancerous.Usually such cells can be characterized by one or more differences fromtheir nearest counterparts that signal the onset of cancer progressionor significant risk for the start of cancer progression. Suchprecancerous changes, if detectable, can usually be treated withexcellent results.

In general, a precancerous state will be associated with the incidenceof neoplasm(s) or preneoplastic lesion(s). Examples of known and likelypreneoplastic tissues include ductal carcinoma in situ (DCIS) growths inbreast cancer, cervical intra-epithelial neoplasia (CIN) in cervicalcancer, adenomatous polyps of colon in colorectal cancers, atypicaladenomatous hyperplasia in lung cancers, and actinic keratosis (AK) inskin cancers. Pre-neoplastic phenotypes and genotypes for variouscancers, and methods for assessing the existence of a preneoplasticstate in cells, have been characterized. See, e.g., Medina (2000) J.Mammary Gland Biol. Neoplasia 5(4):393-407; Krishnamurthy, et al. (2002)Adv. Anat. Pathol. 9(3):185-97; Ponten (2001) Eur. J. Cancer Suppl8:S97-113; Niklinski, et al. (2001) Eur. J. Cancer Prev. 10(3):213-26;Walch, et al. Pathobiology (2000) 68(1):9-17; Busch (1998) Cancer Surv.32:149-79.

Gene expression profiles can increasingly be used to differentiatebetween normal, precancerous, and cancer cells. For example, familialadenomatous polyposis genes prompt close surveillance for colon cancer;mutated p53 tumor-suppressor gene flags cells that are likely to developinto aggressive cancers; osteopontin expression levels are elevated inpremalignant cells, and increased telomerase activity also can be amarker of a precancerous condition (e.g., in cancers of the bladder andlung). In one aspect, the invention relates to the treatment ofprecancerous cells. In another aspect, the invention relates to thepreparation of medicaments for treatment of precancerous cells.

In general, a peptide or analog, derivative or mimetic of this inventioncan be used to treat subjects suffering from any stage of cancer (and toprepare medicaments for reduction, delay, or other treatment of cancer).Effective treatment of cancer (and thus the reduction thereof) can bedetected by any variety of suitable methods. Methods for detectingcancers and effective cancer treatment include clinical examination(symptoms can include swelling, palpable lumps, enlarged lymph nodes,bleeding, visible skin lesions, and weight loss); imaging (X-raytechniques, mammography, colonoscopy, computed tomography (CT and/orCAT) scanning, magnetic resonance imaging (MRI), etc.); immunodiagnosticassays (e.g., detection of CEA, AFP, CA125, etc.); antibody-mediatedradioimaging; and analyzing cellular/tissue immunohistochemistry. Otherexamples of suitable techniques for assessing a cancerous state andeffective cancer treatment include PCR and RT-PCR (e.g., of cancer cellassociated genes or “markers”), biopsy, electron microscopy, positronemission tomography (PET), computed tomography, magnetic resonanceimaging (MRI), karyotyping and other chromosomal analysis,immunoassay/immunocytochemical detection techniques (e.g., differentialantibody recognition), histological and/or histopathologic assays (e.g.,of cell membrane changes), cell kinetic studies and cell cycle analysis,ultrasound or other sonographic detection techniques, radiologicaldetection techniques, flow cytometry, endoscopic visualizationtechniques, and physical examination techniques.

In general, delivering a peptide or analog, derivative or mimetic ofthis invention to a subject (either by direct administration orexpression from a nucleic acid) can be used to reduce, treat, prevent,or otherwise ameliorate any aspect of cancer in a subject. In thisrespect, treatment of cancer can include, e.g., any detectable decreasein the rate of normal cells transforming to neoplastic cells (or anyaspect thereof), the rate of proliferation of pre-neoplastic orneoplastic cells, the number of cells exhibiting a pre-neoplastic and/orneoplastic phenotype, the physical area of a cell media (e.g., a cellculture, tissue, or organ) containing pre-neoplastic and/or neoplasticcells, the probability that normal cells and/or preneoplastic cells willtransform to neoplastic cells, the probability that cancer cells willprogress to the next aspect of cancer progression (e.g., a reduction inmetastatic potential), or any combination thereof. Such changes can bedetected using any of the above-described techniques or suitablecounterparts thereof known in the art, which typically are applied at asuitable time prior to the administration of a therapeutic regimen so asto assess its effectiveness. Times and conditions for assaying whether areduction in cancer has occurred will depend on several factorsincluding the type of cancer, type and amount of peptide, relatedcomposition, or combination composition being delivered to the host.

The methods of the invention can be used to treat a variety of cancers.Forms of cancer that may be treated by the delivery or administration ofa peptide or analog, derivative or mimetic of this invention andcombination therapies containing the same include squamous cellcarcinoma, leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma,non-Hodgkins lymphoma, hairy cell lymphoma, Burketts lymphoma, acute orchronic myelogenous leukemias, promyelocytic leukemia, fibrosarcoma,rhabdomyoscarcoma, melanoma, seminoma, teratocarcinoma, neuroblastoma,glioma, astrocytoma, neuroblastoma, glioma, schwannomas; fibrosarcoma,rhabdomyoscaroma, osteosarcoma, melanoma, xeroderma pigmentosum,keratoacanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma. The compositions of this invention also can be usefulin the treatment of other carcinomas of the bladder, breast, colon,kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroidor skin. Compositions of this invention also may be useful in treatmentof other hematopoietic tumors of lymphoid lineage, other hematopoietictumors of myeloid lineage, other tumors of mesenchymal origin, othertumors of the central or peripheral nervous system, and/or other tumorsof mesenchymal origin. Advantageously, the methods of the invention alsomay be useful in reducing cancer progression in prostate cancer cells,melanoma cells (e.g., cutaneous melanoma cells, ocular melanoma cells,and/or lymph node-associated melanoma cells), breast cancer cells, coloncancer cells, and lung cancer cells. The methods of the invention can beused to treat both tumorigenic and non-tumorigenic cancers (e.g.,non-tumor-forming hematopoietic cancers). The methods of the inventionare particularly useful in the treatment of epithelial cancers (e.g.,carcinomas) and/or colorectal cancers, breast cancers, lung cancers,vaginal cancers, cervical cancers, and/or squamous cell carcinomas(e.g., of the head and neck). Additional potential targets includesarcomas and lymphomas. Additional advantageous targets include solidtumors and/or disseminated tumors (e.g., myeloid and lymphoid tumors,which can be acute or chronic).

The present invention also provides methods for enhancing anti-cancer oranti-tumor immunity, comprising administering to a subject in needthereof an effective amount of an isolated VISTA antagonist or acomposition containing said isolated VISTA antagonist.

In addition to cancer treatment, the present invention also features amethod of treating a pathogen infection, i.e., a bacterial, viral,parasitic or fungal infection, in a subject or host. This methodinvolves administering or otherwise delivering an effective amount of apeptide or analog, derivative or mimetic of this invention so as toreduce the severity, spread, symptoms, or duration of such infection.Such pathogen infections include, but are not limited to diseases causedby bacteria, protozoa, fungi, parasites, or viruses.

In particular embodiments, a viral infection is treated. Any virusnormally associated with the activity of effector lymphocytes can betreated by the method. For example, such a method can be used to treatinfection by one or more viruses selected from hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-1), herpes simplex type 2 (HSV-2),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papilloma virus, cytomegalovirus (CMV, e.g.,HCMV), echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,measles virus, rubella virus, polio virus, and/or human immunodeficiencyvirus type I or type 2 (HIV-1, HIV-2). The practice of such methods mayresult in a reduction in the titer of virus (viral load), reduction ofthe number of virally infected cells, etc.

In addition to pathogen infections, a peptide or analog, derivative ormimetic of this invention can be administered or otherwise delivered toa subject in association with the treatment of immunoproliferativediseases, immunodeficiency diseases, autoimmune diseases, inflammatoryresponses, and/or allergic responses. Moreover, the invention alsoprovides methods for blocking, inhibiting or neutralizing VISTA-mediatedT cell suppression and/or stimulating an immune response in a subject,comprising administering to the subject in need thereof an effectiveamount of an isolated VISTA antagonist or a composition containing saidisolated VISTA antagonist. Such methods may be useful for treating asubject with a one or more of a bacterial, viral, parasitic and fungalinfections and/or cancer.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Materials and Methods

Peptide Synthesis. AP1049 (SSACDWIKRSCH-amide, wherein Cys4-Cysll form adisulfide bridge; SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His)) and scramblednegative control sequence (SSACKSWRDICH-amide, wherein Cys4-Cysll form adisulfide bridge; SEQ ID NO:2) were prepared using standard Fmoc-basedsolid-phase peptide synthesis (SPPS). The peptides were purified viaHPLC, and analyzed by mass spectrometric using the liquidchromatography-mass spectrometry (LC-MS) and matrix-assisted laserdesorption/ionization (MALDI)

Peptide Discovery Using Phage Display. An M13 phage peptide library wasprovided by Dr. Brian Kay (U. Illinois-Chicago). The VISTA proteinrequired for both the phage display biopanning experiments and theconfirmatory ELISA binding experiments was prepared by conventionalrecombinant protein techniques.

T Cell Proliferation Assay. An VISTA-Ig fusion protein or control Igfusion protein was co-absorbed to a cell-culture plate together with thepolyclonal T cell receptor (TCR) stimuli (i.e., anti-CD3 antibody). Toevaluate the activity of VISTA-specific peptides, peptides (VISTAspecific and scrambled control) were added as a soluble reagent to theculture on day 0, and T cell proliferation and cytokine production wereanalyzed after 3-4 days.

T Cell Priming Assay. VISTA is known to suppress T cell priming whenexpressed on antigen-presenting cells (APCs). VISTA-expressing myeloidAPCs (Cd11b^(hi) MHCII⁺ myeloid cells) were purified from mice spleen,FACS sorted, and irradiated (2500 rads). To test the activity ofVISTA-specific peptides, transgenic T cells such as OT-II werestimulated ex vivo with VISTA-expressing APCs and cognate antigenchicken ovalbumin (15 ng/mL). VISTA-specific peptide or control scramblepeptide was added to the cell culture. T cell proliferation and cytokineproduction was evaluated after 3-5 days of culture. As an additionalspecificity control, VISTA-negative parent cell line A20 or APCspurified from VISTA knockout mice were used.

Foxp3+CD4+ Regulatory T Cell (Treg) Suppression Assay. VISTA plays arole in the suppressive function of Foxp3+CD4+ regulatory T cells(Tregs), as VISTA-blocking monoclonal antibody partially reverses Tregsuppressive activity in the in vitro Treg suppression assay. This assayincludes antigen presenting cells, purified Foxp3+ CD4+ Tregs, andFoxp3− CD4+ naïve T cells, which are stimulated by the polyclonal TCRstimuli. To examine the activity of VISTA-specific peptides, peptides(VISTA specific and scrambled control) were added to the Tregsuppression assay on day 0. T cell proliferation and cytokine productionwere measured on day +3.

Model of Experimental Autoimmune Encephalomyelitis (EAE), a MurineAutoimmune Inflammatory Disease Model for Human Multiple Sclerosis. Ithas been shown that VISTA-blocking monoclonal antibody significantlyaccelerates disease onset, as well as exacerbates disease severity in apassive transfer EAE model. In this model, MOG-specific encephalitogenicCD4+T cells are first primed in donor mice upon immunization with MOGpeptide, and then purified and ex vivo expanded in the presence of MOGpeptide and cytokines (IL23, TGFβ, IL6 and IL1b). Expandedencephalogenic CD4+ T cells are transferred into naïve recipients toinduce disease. To evaluate the activity of VISTA-specific peptides,peptides (VISTA specific and scrambled control) are administered viaintraperitoneal injections to mice either prophylactically (startingfrom day 2) or therapeutically (starting from day +7 during diseaseonset), and continuously every 2 days for the entire duration of theexperiment. Disease severity is evaluated according to the establishedprotocol.

Murine Tumor Models. It has been demonstrated that VISTA suppressestumor-specific T cell responses. VISTA blockade via VISTA-specificmonoclonal antibody significantly enhances anti-tumor immune responsesand inhibits tumor progression in murine tumor models such as the B16melanoma model. The activity of VISTA-specific peptides can be evaluatedin vivo using this tumor model. Mice are inoculated on the flank withB16 tumor cells (15,000 cells) on day 0. Peptides (VISTA specific andscrambled control) are administered via intraperitoneal injections tomice either prophylactically (starting from day 2) or therapeutically(i.e., when tumors are palpable), and continuously every 2 days for theentire duration of the experiment. Tumor growth is measured every 2-3days with a caliper.

Example 2 Enhancement of T Cell Proliferation

VISTA⁺CD11b⁺ monocytes were enriched from naïve splenocytes using CD11bmagnetic beads (Miltenyi). VISTA⁺CD11b^(hi) MHCII⁺ myeloid APCs wereFACS sorted, irradiated (2500 rads), and used as antigen-presentingcells to stimulate OT-II transgenic CD4⁺ T cells in the presence of OVApeptide. Control-Ig, monoclonal antibody specific for VISTA and PD-L1(30 μg/mL), or VISTA-specific peptide (100 μg/mL) were added asindicated. Cell proliferation was measured by tritium incorporationduring the last 8 hours of a 72-hour assay. This analysis indicated thatT cell proliferation was enhanced in the presence of VISTA or PD-L1neutralizing monoclonal antibodies, or the AP1049 peptide (FIG. 1). Infact, the AP1049 peptide stimulated T cell proliferation much betterthan either of the monoclonal antibodies, indicating that the peptidepossesses strong antagonistic activity against VISTA.

Example 3 Enhancement of Anti-Tumor Immunity

Immunogenic bladder carcinoma tumors (MB49) were inoculated in femalemice. AP1049 was tested for its ability to slow tumor growth and/orfacilitate tumor regression. The readout for this assay was tumorgrowth.

MB49 tumors were inoculated in female mice (300k) via intradermal (i.d.)inoculation, which facilitates measurement of tumor size. Mice weretreated with either PBS (control) or VISTA antagonist peptide (AP1049),via daily injections around tumor mass starting on day+1 and continuingfor 2 weeks. Tumor size was measured by caliper every 2-3 days.

Using these methods slowed tumor growth and/or tumor regression in micetreated with AP1049 was obtained as compared with mice treated withcontrol.

As shown in FIG. 2, AP1049 treatment reduced tumor growth in the MB49tumor model, indicating that the peptide may bind to the critical/activesite of VISTA and block the immune-suppressive function of VISTA.

1. An isolated VISTA antagonist comprising a peptide which is identicalto the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer,conjugate, analog, derivative or mimetic thereof.
 2. An isolated VISTAantagonist according to claim 1, comprising a peptide which is identicalto the amino acid sequence of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which comprises apeptide having an amino acid sequence that differs from SEQ ID NO:1 byat most 2 amino acid residues, or an multimer, conjugate, analog,derivative or mimetic thereof. 3-5. (canceled)
 6. The isolated VISTAantagonist according to claim 1, wherein the cysteine residues atpositions 4 and 11 of SEQ ID NO:1(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or theircorresponding position in a variant of said peptide, form a disulfidebridge.
 7. An isolated VISTA antagonist according to claim 1, which hasbeen modified to improve binding affinity and/or stability.
 8. Theisolated VISTA antagonist of claim 7, wherein the modification isselected from the group consisting of PEG, acetylation, XTEN, albuminand multimerization.
 9. The isolated VISTA antagonist according to claim1, which is directly or indirectly attached to an immunoglobulinpolypeptide or a fragment thereof.
 10. The isolated antagonist of claim9, wherein said immunoglobulin polypeptide comprises a human IgG1, IgG2,IgG3 or IgG4 constant region or fragment thereof. 11-12. (canceled) 13.An isolated VISTA antagonist according to claim 1, which comprisesanother moiety that targets said peptide to a target site. 14-20.(canceled)
 21. A composition suitable for therapeutic, prophylactic ordiagnostic use comprising a therapeutically, prophylactically ordiagnostically effective amount of the isolated VISTA antagonist ofclaim
 1. 23. The composition of claim 21, further comprising anothertherapeutic agent.
 24. The composition of claim 23, wherein the othertherapeutic agent is an anti-cancer agent, an anti-viral agent, acytokine or an immune agonist.
 25. The composition of claim 21, whereinthe other therapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1or PD-L2 fusion proteins, and EGFR antagonists. 26-32. (canceled)
 33. Amethod for stimulating an immune response in a subject, comprisingadministering to the subject in need thereof an effective amount of anisolated VISTA antagonist according to claim 1 optionally comprisinganother therapeutic agent selected from an anti-cancer agent, ananti-viral agent, a cytokine or an immune agonist.
 34. The method ofclaim 33, wherein the subject has an infection selected from the groupconsisting of bacterial, viral, parasitic and fungal infections. 35-39.(canceled)
 40. The method of claim 33, for treating cancer in a subject.41. The method of claim 33, which is for enhancing anti-cancer oranti-tumor immunity, comprising administering to a subject in needthereof an effective amount of an isolated VISTA antagonist according toclaim 1 and optionally comprising another therapeutic agent selectedfrom an anti-cancer agent, an anti-viral agent, a cytokine or an immuneagonist.
 42. The method of claim 33, which is for treating or preventingcancer, inhibiting tumor invasion and/or cancer metastasis, comprisingadministering to a subject in need thereof an effective amount of saidVISTA antagonist and optionally comprising another therapeutic agentselected from an anti-cancer agent, an anti-viral agent, a cytokine oran immune agonist.
 44. The method of claim 33, which is for treating orpreventing a viral infection, comprising administering to a subject inneed thereof an effective amount of said VISTA antagonist and optionallycomprising another therapeutic agent selected from an anti-cancer agent,an anti-viral agent, a cytokine or an immune agonist. 45-50. (canceled)